Name: sampling and pretreatment of tooth enamel carbonate for stable carbon and oxygen isotope analysis
Uploaded: 2018-08-15T14:30:00
Description: Watch this Scientific Journal Video about Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis at JoVE.com

We would like to thank the Max Planck Society for funding this research as well as the recent setting up of a Stable Isotope laboratory at the Department Archaeology, Max Planck Institute for the Science of Human History.

The following protocol follows the guidelines of the Light Isotope Mass Spectrometry Laboratory at the Max Planck Institute for the Science of Human History. Appropriate ethics permissions from national and international committees should be sought for analyses involving endangered modern or historical faunal specimens, and for the use of archaeological and faunal material of interest to contemporary stakeholders. In this paper, the samples used were archaeological and fossil specimens. No living humans were used in this...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isotopic+generation:+four+decades+of+stable+isotope+analysis+in+African+archaeology.">An isotopic generation: four decades of stable isotope analysis in African archaeology.</a> Azania: Archaeological Research in Africa. 51 (1), 88-114 (2016).</li><li>Ventresca Miller, A. R., Makarewicz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopic+approaches+to+pastoralism+in+prehistory:+Diet,+mobility,+and+isotopic+reference+sets.">Isotopic approaches to pastoralism in prehistory: Diet, mobility, and isotopic reference sets.</a> Isotopic Investigations of Pastoralism in Prehistory. , 1-14 (2018).</li><li>Hollund, H. I., Ariese, F., Fernandes, R., Jans, M. M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+enamel+carbonate+environments+during+fossilization.">Alteration of enamel carbonate environments during fossilization.</a> Journal of Archaeological Science. 26 (2), 143-150 (1999).</li><li>Roche, D., S&#233;galen, L., Balan, E., Delattre, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+assessment+of+Miocene-Pliocene+tooth+enamel+from+Tugen+Hills+(Kenyan+Rift+Valley)+through+FTIR,+chemical+and+stable-isotope+analyses.">Preservation assessment of Miocene-Pliocene tooth enamel from Tugen Hills (Kenyan Rift Valley) through FTIR, chemical and stable-isotope analyses.</a> Journal of Archaeological Science. 37 (7), 1690-1699 (2010).</li><li>Roberts, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calling+all+archaeologists:+guidelines+for+terminology,+methodology,+data+handling,+and+reporting+when+undertaking+and+reviewing+stable+isotope+applications+in+archaeology.">Calling all archaeologists: guidelines for terminology, methodology, data handling, and reporting when undertaking and reviewing stable isotope applications in archaeology.</a> Rapid Communications in Mass Spectrometry. , (2018).</li><li>Snoeck, C., Pellegrini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+bioapatite+carbonate+pre-treatments+for+isotopic+measurements:+Part+1-Impact+on+structure+and+chemical+composition.">Comparing bioapatite carbonate pre-treatments for isotopic measurements: Part 1-Impact on structure and chemical composition.</a> Chemical Geology. 417, 394-403 (2015).</li><li>Pellegrini, M., Snoeck, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+bioapatite+carbonate+pre-treatments+for+isotopic+measurements:+Part+2-Impact+on+carbon+and+oxygen+isotope+compositions.">Comparing bioapatite carbonate pre-treatments for isotopic measurements: Part 2-Impact on carbon and oxygen isotope compositions.</a> Chemical Geology. 420, 88-96 (2016).</li><li>Wright, L. 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The challenges of successful sampling (bulk and incremental) of dentition relies on the access to knowledge regarding drilling techniques and sample preparation, alongside the investment in relatively inexpensive equipment. These challenges are easily surmountable when clear instructions are available concerning&#160;sampling and pretreatment approaches. In this article, we hope to have disseminated these in a clear, concise fashion for researchers new to these methods. Scholars applying these methods for the first time ...

Stable carbon and oxygen isotope analyses of tooth enamel carbonate has been used to study past human dietary intake, weaning, and mobility, as well as faunal reliance on vegetation, the movement of animals, and livestock foddering. These applications have been comprehensively discussed and reviewed for a variety of environmental conditions indicating the effects of local aridity, temperature, water sources, and vegetation compositions1,2,3,4,5,6. The diversity of potential applications in archaeology and paleontology, as well as the good preservation of tooth enamel carbonate, has made it an attractive material for stable isotope work3. Methods of sampling, pretreatment, and diagenesis screening are briefly described in a number of previous publications1,7. However, thorough verbal and visual demonstrations remain largely unavailable, particularly to people outside of archaeological science laboratories and among laboratory groups with limited funding where the interest in the use of this technique is increasing5.
Tooth enamel is primarily made up of hydroxyapatite (bioapatite) crystallites8 larger than those in bone, making it more resistant to post-mortem diagenetic ionic substitutions and contamination3. Modern studies have demonstrated that stable carbon isotope (&#948;13C) measurements of faunal tooth enamel reliably record animal diet and behavior9,10. The stable oxygen isotope (&#948;18O) value of tooth enamel is determined by the oxygen isotopic composition of ingested water, which includes water in plant and animal foods, drinking water, respiration, as well as various environmental impacts on the water which can lead to further isotopic fractionation (e.g., aridity, temperature, altitude, rainfall amount, continental location)11. This has made it a popular method for dietary and environmental reconstruction in archeological, paleoecological, and paleontological research.
The period of tooth enamel formation is relatively short (years) and differs depending on the tooth being sampled. For humans, first molar enamel mineralizes between birth and 3 years of age, premolars mineralize between 1.5 and 7 years of age, second molars mineralize between 2.5 and 8 years of age, and third molars mineralize during adolescence, between 7 and 16 years12. Given that tooth enamel forms incrementally over its period of formation, it can be sampled in bulk along the entire growth axis or sampled sequentially in order to investigate the changes in diet and environment that have occurred during the formation period13. Chronologically-ordered dietary change within a given tooth is observable for humans and other animals1,14, providing information regarding inter-annual seasonal and dietary variation.
While enamel is usually resistant to diagenesis, isotopic modifications resulting from the burial environment are possible and have been observed15,16, making experimental checks and pretreatment choices useful. While it is not the only available method, Fourier transform infrared spectroscopy (FTIR), particularly in Attenuated Transmission Mode, has emerged as a quick, inexpensive, and relatively accessible method for assessing taphonomic alteration in tooth enamel, particularly in paleontological contexts17,18,19,20. However, detailed protocols and recording standards remain relatively inaccessible to many people outside the fields of geochemistry or material science.
Reaction times and the chemicals employed by researchers in the pretreatment of tooth enamel also vary considerably in the literature, often with limited consideration as to what this variability may do to stable carbon and oxygen isotope values of the sample21,22. Here, we report an approach that uses dilute acetic acid (0.1 M) for the pretreatment of enamel powder samples. However, given that the differences in isotopic measurements resulting from pretreatment are relatively minor for tooth enamel, it is best for the researchers to follow the protocols for datasets with which they wish to compare their data to11. Furthermore, where small sequential samples are taken, particularly on Holocene samples, no pretreatment may be chosen (following pilot diagenetic tests) to avoid sample wastage.
Although the methods we report here are by no means new, to our knowledge, this is the first time that a thorough written and visual documentation of bulk and sequential sampling, pretreatment choices, and diagenetic check methods (in the form of FTIR) for tooth enamel have been made widely available to a varied academic audience. While we hope our efforts will make this approach more easily accessible to a wider number of individuals and laboratories, researchers who want to apply and publish this technique must be aware of minimum reporting standards, diagenetic considerations, and presentation requirements overviewed elsewhere20, as well as potential interpretive complexities that will be unique to their study region, taxa analyzed, and time period5.

<table>
	<tbody>
		<tr>
			<td>Dremel Micro</td>
			<td>Dremel</td>
			<td></td>
			<td>https://www.dremel.com/en_US/products/-/show-product/tools/8050-micro</td>
		</tr>
		<tr>
			<td>Diamond-tipped drill bit</td>
			<td>Dremel</td>
			<td></td>
			<td>https://www.dremel.com/en_US/products/-/show-product/accessories/7122-diamond-wheel-point</td>
		</tr>
		<tr>
			<td>1.5 mL micro-centrifuge tube</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/t2422?lang=de&#38;region=DE&#38;gclid=EAIaIQobChMI7pHRpauW2QIV77ftCh1p1wjhEAAYASAAEgKzkvD_BwE</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td></td>
			<td></td>
			<td>Linear Formula:&#160;CH3OH</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td></td>
			<td></td>
			<td>Linear Formula:&#160;CH3CO2H</td>
		</tr>
		<tr>
			<td>Dremel rig set-up (workstation)</td>
			<td>Dremel</td>
			<td></td>
			<td>https://www.dremel.com/en_US/products/-/show-product/tools/220-01-workstation</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>http://www.thermofisher.com/order/catalog/product/75002401</td>
		</tr>
		<tr>
			<td>Mini-centrifuge</td>
			<td>Sprout</td>
			<td></td>
			<td>http://www.heathrowscientific.com/sprout-mini-centrifuge-4</td>
		</tr>
		<tr>
			<td>Freeze drier</td>
			<td>Zirbus Technology</td>
			<td></td>
			<td>http://www.zirbus.com</td>
		</tr>
	</tbody>
</table>

sampling and pretreatment of tooth enamel carbonate for stable carbon and oxygen isotope analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Using the sampling procedure presented above, incremental enamel bioapatite samples were prepared. The analysis of bioapatite in enamel depends on the accuracy of sampling, whether bulk or incremental. In this case, we have chosen to present the results of archaeological samples (two sheep) from different climatic zones. Incremental samples were analyzed from sheep second molars and labelled starting at the ERJ (Figure 4). Incremental sample locations were nu...

Watch this Scientific Journal Video about Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis at JoVE.com

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

This method can answer key questions in archeology, paleontology, and ecology, such as domesticated animal diets, animal management strategies, and animal mobility. The main advantage of this technique is that it is relatively nondestructive and that it is focused on a material that is reasonably resistant to chemical changes in the burial environment. Individuals new to this method will struggle because there are no available visual aids for the sampling of paleontological and archeological remains of humans and animals.Visual demonstration of this method is critical because it is difficult to learn and practice on archeological materials is limited. To begin bulk sampling, set up a handheld drill as outlined in the text protocol, with a clean diamond-tipped drill of even shape, attached via a chuck to the drill setup. Using the drill, clean the outer surface of the tooth by gentle abrasion.Next, tare a 1.5-milliliter microcentrifuge tube on an appropriately accurate balance. Then, place clean aluminum foil or weighing paper under the drill to collect the enamel powder. Use the drill to deliver gentle and even abrasion, creating a groove parallel to the entire growth axis.Be aware that the amount of sample powder drilled will vary depending on the equipment used for analysis, the pretreatment protocol, and the size of the tooth. Collect the resulting enamel powder, and transfer it into a 1.5-milliliter microcentrifuge tube. Record both the sample number and enamel weight in a laboratory notebook, as well as an electronic database.After each tooth is drilled, clean any used drill bits using 0.5-molar hydrochloric acid on a robust tissue. Wash the drill bits again with an organic solvent on a new robust tissue. Clean the drill itself using either a vacuum or compressed air.Once a drilling session is complete, thoroughly clean the workspace using either a dedicated vacuum cleaner or a dustpan and brush. Wipe down the sampling space with methanol. Clean the drill using either the vacuum or compressed air.For sequential sampling, first clean the surface of the tooth by removing a very thin layer of outer tooth enamel. Choose an appropriate drill bit for the sample, as the width of the sampling line is determined by the diameter of the drill bit. To begin drilling, press the tooth enamel against the drill bit and apply pressure.Drill along the buccal surface perpendicular to the growth axis of the tooth, running from the enamel-root junction to the top of the crown. Make sure to drill from the apex to the base of the crown at the location of the enamel-root junction, resulting in a groove across the enamel layer. After the drilling is complete, weigh the sample to ensure there is enough enamel powder, and record the weight.Repeat the drilling process several times per tooth, with each consecutive line parallel to the previous sample line and about one to two millimeters apart. After this, use calipers to measure the distance of each sampling line from the enamel-root junction. Record each distance for comparison.To begin, use a pipette to add 0.1-molar acetic acid to each sample at a concentration of 0.1 milliliters per one milligram of enamel. Agitate the samples either by shaking or by using an agitator. Let the agitated samples sit for 10 minutes.Centrifuge the samples at 13, 700 g for two minutes. Using a clean pipette, replace the acetic acid with one milliliter of ultrapure water, and agitate either by shaking or by using an agitator. Centrifuge again at 13, 700 g for two minutes.Repeat the process of replacing the ultrapure water and centrifuging two additional times. Test the samples for neutrality. If the sample has not reached neutrality, repeat the washing process until it does.Then, use a pipette to remove the remaining fluid without disturbing the supernatant. Place a precut one centimeter by one centimeter square of Parafilm over each microcentrifuge tube. Using a sharp object, make a small hole in the Parafilm to ensure that the sample dries appropriately, and place the tubes in a freezer overnight.The next day, transfer the samples to the freeze dryer to remove any remaining fluid. Once the samples are dry, remove the Parafilm, and close the microcentrifuge lids. Check the tube labeling, and remark the tubes as necessary.In this study, incremental enamel bioapatite samples are prepared. Each incremental sample location is numbered, and the distance between each location and the enamel-root junction is measured. Diverse carbon and oxygen stable isotope results from two representative sheep confirm that these animals lived in different environments.Sheep A is from a tropical grassland and shows incremental oxygen 18 in a narrow range, between 3.3 and 5.1 per mil. This suggests ingestion of water from sources with similar isotopic values and a lack of strong seasonal shifts in precipitation. In contrast, sheep B, which is from a temperate dry steppe grassland, has oxygen 18 values with a high amplitude of variation, ranging from negative 5.2 to negative 13.1 per mil.This indicates strong seasonal variation in precipitation. Stable carbon isotope values suggest that sheep A's diet consists mainly of C4 plants, while sheep B's diet consists mainly of C3 vegetation. Incremental oxygen 18 and carbon 13 values for a human tooth from a rainforest environment are highly constrained.These values, which fall within a two per mil range, suggest a lack of variation in foraging strategies over the period of enamel mineralization. While attempting this procedure, it is important to remember to practice sequential sampling on modern teeth before drilling ancient samples that are not replaceable. Sequential sampling may take several hours of practice before proficiency is reached.For ancient samples, it is important to choose teeth that can withstand the sampling process. Teeth that are poorly preserved or are liable to crumble are unlikely to prove useful for detailed sequential sampling. Following this procedure, other methods such as strontium isotope analysis can be attempted in order to understand human and animal mobility.After its development, this technique paved the way for researchers in the fields of archeology and paleontology to explore animal herding and migration strategies across diverse regions and time periods. Don't forget that working with drills and acetic acid can be extremely hazardous, and precautions such as googles, gloves, and face masks should always be taken while performing this procedure.

sampling-pretreatment-tooth-enamel-carbonate-for-stable-carbon-oxygen

Research

JoVE Journal

Biochemistry

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be used to model diseases that are otherwise difficult or impossible to study in humans or other animal models. The gut microbiome can have a profound impact on human health and disease. However, the murine gut microbiome is very different than the one found in humans. &#160;There is a need for improved pre-clinical hu-mice models that have an engrafted human gut microbiome. Therefore, we created double hu-mice that feature both a human immune system and stable human-like gut microbiome. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are one of the best animals for humanization due to their high level of immunodeficiency. However, germ-free NSG mice, and various other important germ-free mice models are not currently commercially available. Further, many research settings do not have access to gnotobiotic facilities, and working under gnotobiotic conditions can often be expensive and time consuming. Importantly, germ-free mice have several immune deficiencies that exist even after the engraftment of microbes. Therefore, we developed a protocol that does not require germ-free animals or gnotobiotic facilities. To generate double hu-mice, NSG mice were treated with radiation prior to surgery to create bone-marrow, liver, thymus-humanized (hu-BLT) mice. The mice were then treated with broad spectrum antibiotics to deplete the pre-existing murine gut microbiome. After antibiotic treatment, the mice were given fecal transplants with healthy human donor samples via oral gavage. Double hu-BLT mice had unique 16S rRNA gene profiles based on the individual human donor sample that was transplanted. Importantly, the transplanted human-like microbiome was stable in the double hu-BLT mice for the duration of the study up to 14.5 weeks post-transplant.

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

Stable Isotope Tracing &amp; LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

13C4-Aspartate and 13C5-Glutamine Stable Isotope Tracing, LC-MS Analysis, and SDH Activity Measurement

13C4-Aspartate and 13C5-Glutamine Stable Isotope Tracing, LC-MS Analysis, and SDH Activity Measurement  

To begin, seed the 143B human osteosarcoma cells in a six-well plate. Once cells become 75%confluent, replace the medium in each well with the medium containing 10 millimolar 13C4 aspartate, and incubate for eight hours to achieve a steady state for labeling the cells. For isolating metabolites, cool the prepared buffer overnight at minus 80 degrees Celsius or place it on dry ice.Meanwhile, take a plate from the incubator and aspirate the medium from each well using a pipette. Wash it with PBS twice, and then remove the residual PBS before proceeding further. Now place the plate on dry ice and add 800 microliters of prepared buffer to each well.To facilitate cell lysis, incubate the plate for 15 minutes at minus 80 degrees Celsius. After incubation, scrape each well on dry ice using a cell lifter, and transfer the lysate to a 15 milliliter microcentrifuge tube placed on dry ice. Vortex the lysate for 10 minutes at four degrees Celsius, and centrifuge it at four degrees Celsius and 17, 000 g for 10 minutes.Then transfer the supernatant to a 1.5 milliliter microcentrifuge tube and dry it in a four degree Celsius vacuum concentrator with a cold trap under a high vacuum for approximately six hours. Store the dried metabolite pellet at minus 80 degrees Celsius until further use. For analyzing the sample using liquid chromatography mass spectrometry or LCMS, add 100 microliters of HPLC grade water to the dried pellet and vortex as demonstrated.Centrifuge the samples at four degrees Celsius for 10 minutes at maximum speed. Then transfer 25 microliters of supernatant to the LCMS vial, and inject two microliters into the LCMS system. To perform liquid chromatography, use a constant flow rate of 0.15 milliliter per minute, under the gradient mode of a mobile phase B from 80 to 20%for 20 minutes, 20 to 80%for 0.5 minutes, and hold at 80%for 7.5 minutes against mobile phase A.Next, perform mass spectroscopy by choosing a full scan between mz 70 to 1, 000 dalton.And resolution at 70, 000. Set AGC target of one times 10 to the sixth, and a maximum injection time of 20 milliseconds. Next, set the runtime to 16.5 minutes.Polarity set to positive. AGC target set to one to the power of five. Maximum IT set to 20 milliseconds.And scan range set to 70 to 1, 000 mass-to-charge ratio. Then set the spray voltage at 3.0 kilovolts. And the heated capillary at 275 degrees Celsius.Select the sheath gas flow at 40 units, the auxiliary gas flow at 15 units, and the sweep gas flow at one unit. For 13C5 glutamine isotope tracing, once the cells reach 75%confluency, change the medium to two millimolar 13C5 glutamine-containing medium, and incubate to achieve a steady state of labeling the cells of interest. Isolate and analyze the metabolites as previously demonstrated.After isolating and analyzing the metabolites, analyze the succinate dehydrogenase or SDH activity by calculating the percent labeling of 13C3 fumarate, 13C4 fumarate, 13C3 succinate, and 13C4 succinate. Then evaluate succinate oxidation and fumarate reduction using the formula. In vehicle-treated conditions, the SDH complex favored the forward activity, and the incorporation of 13C4 succinate into 13C4 fumarate was higher than 13C3 fumarate into 13C3 succinate.In antimycin-treated osteosarcoma cells, the SDH complex favored the reverse activity, and the incorporation of 13C3 fumarate into 13C3 succinate was more significant than 13C4 succinate into 13C4 fumarate.